Isotope labeling methods

ABSTRACT

The present invention relates to a method for the analysis of differential expression of proteins employing a radioactive label, characterized by cleaving a tag from peptides labeled with a cICAT reagent, separating and purifying the resultant labeled peptides, and performing an analysis in mass spectrometry.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to an isotope labeling method for the analysis of differential expression of proteins. Specifically, the present invention relates to an improved method for performing an analysis of differential expression of a plurality of small-amount proteins in samples employing an ICAT reagent containing a cleavable tag (which hereinafter is simply referred to at times as a “cICAT reagent”), and to a system for such an analysis.

2. Description of Related Art

Genome analysis has actively been conducted in connection with diseases and aging and gives rise to a lot of results. Recently, further advancing of the analysis has made attempts to analyze a population of proteins which are expression products of genes in diseased or aging tissues and normal tissues (proteosome), thereby to identify proteins involved in diseases and aging. Various methods for the analysis of differential expression have been developed and are used for analyzing these proteosomes. It is on isotope labeling methods that attention is focused among them.

Isotope labeling method are an analytical method by which two types of isotope-labeled reagents that specifically react with amino acids or others in a protein (light- and heavy-chain labeled reagents designed to have a difference only in mass number employing an isotope) are used to separately label respective proteins to be compared, followed by trypsin treatment or the like, and the resulting peptides are subjected to measuring the ratio of amounts of light- and heavy-chain labeled peptides on a mass spectrometer, thereby to quantitatively examine differential expression of proteins. It is likely that these methods can be employed to identify proteins associated with diseases, for example, by performing an analysis of differential expression between proteins from patients and healthy individuals.

There are provided ICAT reagents as means for improving quantitativity, reproducibility, and other properties in these isotope-labeling methods. A cICAT reagent, which is a type of isotope-labeled reagents that specifically react with particular sites in a protein, is designed such that its segment contains a tag and labeled peptides containing the tag can be purified specifically, for example, on affinity columns, and in addition, the tag moiety can be cleaved from the labeled peptides, for example, with acid treatment (Hansen, K. C. et al., Mol. Cell Proteomics, 2:299-314, 2003). For example, there is a known routine procedure which employs a cICAT reagent using biotin as the tag (ABI protocol), and there are many reports saying that this protocol is effective in making a precise analysis of differential expression of many proteins in a variety of tissues and cells (T. Toda, et al., Eds., In Frontier of Disease Proteomics Idenshi, Igaku MOOK 2 (ISSN 1349-2527), pp. 233-243, 2005 (published by Medical Do), in Japanese). However, there have been few reports on the results of analyses of differential expression of proteins, which were performed in accordance with the above-described routine procedure, in samples, such as serum, having a plurality of small-amount proteins. Only twenty to thirty of serum proteins were identified and quantified (Zieske, L. R. et al., ASMS 2003, Poster Number W-032).

As mentioned above, isotope labeling methods utilizing cICAT reagents which are previously known are not always effective when making an analysis of differential expression of proteins in samples having a plurality of small-amount proteins, and thus there is great need of methods which are more effective for the analysis of differential expression. A purpose of the present invention is to provide, by improving an isotope labeling method employing a cICAT reagent, a method which effectively makes an analysis of differential expression of a plurality of small-amount proteins present in a sample, and is to provide a system therefor.

SUMMARY OF THE INVENTION

The present inventors have made extensive studies in view of the above-described circumstances, and in consequence have found that samples in which, according to a routine procedure, serum samples were treated with a cICAT reagent and labeled peptides containing the tag were fractionated and purifying, followed by tag cleaving treatment of the obtained tag-containing sub-fractions, contained large amounts, which were not expected, of the tag and tag-containing byproducts derived from the reagent (which are collectively referred to as the “tag and others”), and these remaining tag and others are responsible for significantly reducing the number of serum proteins to be identified and quantified. Thus, the inventors modified the routine procedure and in consequence, have found that it is possible to perform an analysis of a much larger number of small-amount proteins than with the routine procedure, when the tag portion of the cICAT labeled peptides is cleaved in advance and the resulting sample is loaded on a column to move the remaining tag and others, followed by analyzing, on a mass spectrometer, the labeled peptides obtained by the separation and purification of the labeled peptides, leading to the completion of the invention.

Therefore, the present invention provides the following:

(1) a method for the analysis of differential expression of proteins employing isotope labeling, characterized by cleaving a tag from peptides labeled with a cICAT reagent, separating and purifying the resultant labeled peptides, and performing an analysis in mass spectrometry;

(2) the method according to (1), wherein the step of separation and purification is carried out using column chromatography and wherein the removal of the tag and others and the separation and purification of the cICAT-labeled peptides are carried out concurrently;

(3) the method according to (1) or (2), wherein the tag is biotin;

(4) the method according to any one of (1) to (3), wherein the peptides are derived from serum proteins;

(5) a system for performing an analysis of differential expression of small-amount proteins in a sample, characterized by employing a method according to any one of (1) to (3); and

(6) the system according to (5), wherein the sample is a serum sample.

According to the present invention are provided methods and systems enabling one to perform an efficient analysis of differential expression of a plurality of small-amount proteins in samples. Such methods can be used, for example, to make an analysis of differential expression between serum proteins from patients and healthy individuals, which has utility, for example, in searching proteins associated with diseases, and other applications.

The present invention, which provides methods and systems enabling one to perform an efficient analysis of differential expression of a plurality of small-amount proteins present in samples, can be used in the fields of proteomics studies, analytical instruments, and others,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows biotin fractions from a sample in which biotin-bound, cICAT-labeled serum peptides have been TFA treated and a fraction pattern by an SCX column chromatography of the cICAT-labeled serum peptides having the biotin removed therefrom. The peak of the biotin can be seen at a retention time of about 5 minutes and the peak of biotin-containing byproducts derived from the reagent at a retention time of about 14 minutes, demonstrating that the separation of the peptide peaks from the byproduct peak has been achieved.

FIG. 2 shows a Venn diagram representation of top 119 human-serum proteins identified by Q-Star XL and by ABT-4700.

FIG. 3 shows a Venn diagram representation of all the 311 human-serum proteins identified by Q-Star XL and by ABI-4700.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention will now be described in detail and unless otherwise explained, the terms as used herein are intended to have the meaning usually that are understood in the art.

The present invention, in a first aspect, provides a method for the analysis of differential expression of proteins employing isotope labeling, characterized by cleaving a tag from peptides labeled with a cICAT reagent, separating and purifying the resultant labeled peptides, and performing an analysis in mass spectrometry. Protein containing samples which can be subjected to the method according to the present invention are not limited in particular, and any sample may be used, including samples derived from animals, plants, and microorganisms. Examples of protein containing samples derived from animals include samples of body fluids obtained from mammals, particularly, from humans, such as serum, saliva, urine, sweat, and others. Examples of samples derived from plants include fruit juices, extracts of stems and leaves, extracts of seeds, extracts of underground stems, and others. Samples derived from microorganisms include various fermentations, cultures, microbial homogenates, and others. The present invention can be applied to these samples containing proteins, thereby to enable one to made an analysis of differential expression of the proteins, so as to investigate metabolic mechanisms of organisms, including animals, plants, and microorganisms. In particular, the present invention can be used to carry out proteomic studies, for example, for the identification of proteins associated with animal diseases and aging, or alternatively, for example, to make a diagnosis or examination of diseases in animals, including humans. As demonstrated in Examples, the present invention displays its power, especially in the analysis of differential expression of a wide variety of small-amount proteins in serum.

In the present method for the analysis of differential expression of proteins, protein containing samples described above are first treated with a cICAT reagent to obtain cICAT-labeled proteins. Conditions for the reaction of the cICAT reagent and the proteins contained in a sample will be varied, depending on the type of amino acids in the proteins to be labeled and the properties of the cICAT reagent. In general, the cICAT reagent is comprised of a site at which the reagent binds to a protein (for example, a site at which the reagent binds to cysteine of a protein), an isotope-labeled linker, a tag-cleaving site, and a tag. Binding of the cICAT reagent and a protein is usually covalent. As the isotope, various isotopes can be used, and stable isotopes are preferable. For example, combinations of ¹H and ²D, ¹²C and ¹³C, and others are employed. It may be possible that a sample from normal tissues is labeled with a ¹²C-containing cICAT reagent and a sample from diseased tissues is labeled with a ¹³C-containing cICAT reagent, thereby to perform an analysis of differential expression of proteins. As the tag, tags of any type can be used if their attachment facilitates the separation and purification of peptides and does not exert detrimental effects on the analysis of peptides, and include, for example, sugar containing groups, and others. Biotin is preferably used as the tag, because of easy and specific purification by use of avidin affinity chromatography. As the tag-cleaving site, sites of any type can be used if the tag can be cleaved with ease and without exerting detrimental effects on the labeled peptides. For example, use is usually made of tags which can be cleaved easily with acid treatment, such as TFA (trifluoroacetic acid).

It is well known in the art that an “ICAT” reagent stands for “Isotope-Coded Affinity Tags.” In the specification, an ICAT reagent containing a cleavable tag is referred to as a cICAT reagent, as described above. As cICAT reagents for use in the present invention are included various reagents, and they are commercially available. Typically, there is a Cleavable ICAT reagent from ABI employing biotin as the tag, which is preferably used in the present invention. The “Cleavable ICAT” is the registered trade name of ABI.

After the reaction of the proteins in a sample and the cICAT reagent, the resulting cICAT-labeled proteins are subjected to proteolysis to obtain cICAT-labeled peptides. This proteolysis can be carried out in various ways. For example, acid hydrolysis, enzymatic hydrolysis, and others can be utilized, Preferably, enzymatic hydrolysis is employed. Preferable proteolytic enzymes include trypsin, pepsin, and others, and trypsin is used more preferably.

After that, the tag portion is cleaved from the cICAT-labeled peptides obtained as described above. The cleavage of the tag at this stage is a feature of the present invention. In order to concentrate the cICAT-peptides and remove the contaminating materials, the cICAT-labeled peptides may be purified prior to the tag cleavage. To this end, it is usual to employ affinity chromatography using a substance which can specifically bind to the tag. For example, when the tag is biotin, column chromatography using a resin to which avidin has been bound can be performed, thereby to collect the cICAT-labeled peptides. Methods for cleaving the tag portion from the cICAT-labeled peptides will be varied, depending on the structure of the cICAT reagent, in particular, the type of tags, the class of analytes, and others. The cleavage reaction must be carried out under conditions exerting no effect on the peptides to be analyzed. For example, in the case of using a Cleavable ICAT reagent from ABI, TFA can be employed to cleave the biotin tag.

In the case when a subsequent step of separation and purification is carried out without the cleavage of the tag at such a stage as described above (i.e. in the case of conventional procedures, for example, when the ABI protocol is used), large amounts of the tag and others remain in the obtained sample, resulting in significant interference in the identification and quantification of proteins, especially small-amounts proteins. Moreover, conventional procedures require applying tag cleavage treatment to each of the peptide fractions obtained from the step of separation and purification and then carrying out the step of mass spectrometry, and thus take much time and labor. In contrast, the method of the present invention is free from these disadvantages and allows an efficient identification and quantification of a wide variety of small-amount proteins in a sample.

Subsequently, samples of the labeled peptides obtained by cleaving the tag according to the method of the present invention are subjected to the step of separation and purification. Although the step of separation and purification can be carried out using various procedures, it is preferable that column chromatography is employed so that the removal of the tag in the sample and the separation and purification of the peptides are carried out concurrently. Various supports for chromatography are commercially available and can be selected as appropriate, depending on the type of tags and analytes. For example, silica gel-based supports may be used, or SCX supports (poly-LC-sulphoethyl A supports) may be used, or supports having affinity for avidin (when the tag is biotin) may be used. Column conditions for elution will be determined as appropriate, depending on the properties of analytes and tags, and others. It may be effective to employ salt concentration gradient elution methods. It is preferable in terms of resolution, rapidity, and others that column chromatography is carried out using HPLC. In addition, the step of separation and purification is not limited to the use of columns, and methods of using filters, batch processes, and others can be employed. Such a step of separation and purification may be carried out twice or more. Furthermore, samples may be concentrated before subjecting them to the step of separation and purification. In general, chromatograms are recorded and fractions corresponding to respective peaks are pooled in the step of separation and purification. Each of the fractions can be desalted and then subjected to mass spectrometry.

The peptide fractions which are obtained in this way from the step of separation and purification are subjected to mass spectrometry (MS) to identify proteins in the sample. Various procedures and methods for performing MS measurements are known and many instruments therefor are commercially available, so that selection can be made as appropriate to use them. Additionally, in order to seek improvements in performance of separation and qualitative determination, analytical procedures have been developed which combine gas chromatography (GC) or liquid chromatography (LC) with MS (GC/MS, LC/MS, LC/MS/MS, and the like), and many instruments for those procedures are commercially available. In particular, LC/MS is suitable for analysis of proteins and peptides as in the present invention. In the specification, not only mere MS, but also configurations incorporating MS, such as GC/MS, LC/MS, and LC/MS/MS are referred to as mass spectrometry (MS). Ionization methods in MS usually use electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption ionization (MALDI) methods, and methods for analyzing ionized fragments include, for example, ion trap, time of flight, quadrupole, Fourier transform, and other methods, and thus selection can be made as appropriate to use them.

As described above, the method of the present invention is a method in which the tag is cleaved in a lump prior to the separation and purification of the labeled peptides, and therefore does not require applying, as in the conventional procedures, each of the peptide fractions which are obtained in the step of separation and purification to tag cleavage treatment, and thus can save time and labor. In addition, the method of the present invention is a method suitable for the identification/quantification of a wide variety of small-amount proteins in samples. Accordingly, the method of the present invention is suitable for a high throughput analysis of a wide variety of small-amount proteins in samples. Therefore, the present invention, in a further embodiment, provides a system for performing an analysis of differential expression of small-amount proteins in samples, the system characterized by employing the method of the present invention as described above. The system of the present invention is suitable, for example, for an analysis of differential expression, preferably a high throughput analysis, of proteins in serum samples of mammalian animals, in particular, of humans.

The present invention will now be described specifically and in detail by way of examples, which are intended to be only illustrative of the present invention and not to be limiting of the scope of the present invention.

EXAMPLES

1) Removal of major proteins in serum by an Agilent antibody column:

A serum fraction which was obtained by employing an Agilent antibody column (for the removal of albumin, IgG, α1-antitrypsin, IgA, transferin, and haptoglobin, 10×100 mm) to remove the six major serum proteins described above was used for analysis. Accordingly, 200 μl of human serum (Rockland Immunochemicals, Inc.) was centrifuged at 15,000 rpm, diluted 5 times in Agilent Binding Buffer A, filtered through a 0.22 μm filter, and loaded onto the above-described antibody column to collect the flow-through fraction in which the six major proteins described above had been removed on the above-described antibody column. The flow-through fraction was concentrated and buffer changed on a Centriprep centrifugation filter unit (YM-3, Millipore) to 50 mM Tris/HCI, 0.1% SDS (pH 8.5), followed by determining the protein concentration by Lowry method.

2) cICAT reaction of human normal serum:

The serum protein faction in which the six major serum protein described above had been removed (a final concentration of 1 mg/ml) was solubilized in 50 mM Tris/HCl, 0.1% SDS (pH8.5), reduced with TCEP (a final concentration of 1 mM; at 95° C. for 10 min.), and then reacted with 2.2 mM of a Cleavable ICAT reagent (Applied Biosystem (ABI), ¹³C (H chain) or ¹²C (L chain) label) at 37° C. for 2 hours. An unreacted reagent was quenched with 1.0 mM TCEP, and the H-chain and L-chain samples were mixed at an equal amount and subjected to digestion with trypsin (Promega, TPCK treated) at 37° C. for 16 hours. The resultant digestion was loaded onto an SCX column (poly-LC-sulphoethyl A column (4.6×100 mm)) using a Vision Workstation system (ABI). After adsorption and washing in 10 mM KH₂PO₄, pH 2.8, 25% CH₃CN (SCX binding buffer), elution was carried out with the SCX binding buffer plus 0.5 M KCl (SCX elution buffer). The eluted fraction was applied to a large avidin-column (6.2×66.5 mm), the flow-through portion was washed, and the adsorbed cICAT-reagent-reacted peptides were eluted with 30% CH₃CN/0.4% TFA (using the Vision Workstation System) . The eluted fraction was dried and then reacted with 95% TFA (containing 5% scavenger) at 37° C. for 2 hours to cleave the biotin segment to obtain the ICAT-labeled peptides (H and L chains). The reaction mixture containing these peptides was subjected to dryness under reduced pressure, and then dissolved in the SCX binding buffer. The peptide solution was applied again to an SCX column, which was washed thoroughly with the SCX binding buffer to remove fractions of the tag and others. After that, the SCX binding buffer plus KCl (gradient of 0 to 0.5 M) was used to fractionate the peptides (50 fractions) (FIG. 1). Each of these fractions was desalted on a C18 trap column and subjected to dryness under reduced pressure.

3) Separation and purification of cICAT-peptides by nano-LC:

The ICAT-labeled peptides which were fractionated and desalted by SCX were re-dissolved in 0.1% TFA-2% CH₃CN and analyzed on nano-LC (LC-Packings)/Q-Star XL (ABI, ESI-Q/TOF, hereinafter referred to as “Q-Star”) and on nano-LC/Probot (LC-Packings)/ABI-4700 Proteomics Analyzer (ABI, MALDI-TOF/TOF, hereinafter referred to as “ABI-4700”) (column: PepMap™ C18 100, 3 μm, 100 angstroms, 75 μm (i.d.)×150 mm (LC-Packings), mobile phase for Q-Star: a linear gradient of A: 5% CH₃CN/0.1% HCOOH and B: 95% CH₃CN/0.1% HCOOH, mobile phase for ABI-4700: a linear gradient of A: 5% CH₃CN/0.1% TFA and B: 95% CH₃CN/0.1% TFA). Each mass spectrometry was performed as follows.

4) Measurements on Q-Star (ESI-Q/TOF):

A BSA digestion (50 fmol) was used to adjust the nano-LC. After confirming that a predetermined sequence coverage (a degree of about 40%) was achieved, measurements of samples were made according to the routine procedure. Measurements were made in an automatic measurement mode (IDA mode) in which one cycle is of a total of 7 seconds: MS for 1 second, 1st MS/MS for 3 seconds, and 2nd MS/MS for 3 seconds.

5) Measurements on ABI-4700 (MALDI-TOF/TOF):

A sample was separated on the nano-LC/Probot system and spotted with a matrix (CHCA, 875 ng/well). A sample plate was inserted into the apparatus, and then the laser intensity was determined on an MS reflector mode for the measurement of calibrants (Des-[Argl]-bradykinin (M+H)⁺=904.468; angiotensin I (M+H)⁺=1296.685; ACTH (1-17) (M+H)⁺=2093.087; ACTH (18-39) (M+H)⁺=2465.199; ACTH (7-38) (M+H)⁺=3657.929). Subsequently, some of the spots where the sample was applied were randomly selected and the laser intensity was determined for MS and MS/MS measurements. After that, a method for automatic measurements was prepared and MS-MS/MS sequential measurements were made (MS accumulations: 1250, MS/MS accumulations: 2000).

6) Results of the analysis of human serum protein by the cICAT method:

The date obtained by the above-described analytical instruments for mass spectrometry were analyzed employing a combined data identification system (HiSpec) using RefSeq as the DB to be searched, and peptides and proteins were identified and the H and L chains were comparatively quantified. Since the H-chain and L-chain labels were allowed to be reacted at an equal amount (as described above), the ratio of H-chain labeling and L-chain labeling would theoretically be 1. Results are shown in Table 1, which ranks identified protein in decreasing order of total score (Rank, Q-Star or ABI-4700) and summarizes their generic names (Description), GI numbers, molecular weights (Mass), score values of the H and L chains, ratios of the H/L chains (Ratio, comparative quantification value), the number of Cys residues (Total cys), the number of trypsin-digestion fragments actually identified of the H- and L-chain labeling reactions (NRPepCnt (H, L)), and sequence coverages (Protein Coverage (H, L)).

H factor 1 (complement); H factor-1

139125.4

0.92 0.97

1563.5 933.8

30 25 (complement); complement factor H; factor H-like 1; H factor 2 (complement)

plastinogen

90689

0.95 0.96

1159.5 882.6

34 33

coagulation factor II precursor;

70036.9

0.98 0.98

959.7 707.5

32 35

alpha 2 macroglobulin precursor

163278

0.92 0.95

1222.9 884.8

15 15

complement component 3 precursor

187164.1

0.92 0.88

993.4 832.1

11 10 a cylation-stimulating protein cleavage product

transferrin; PRO2086 protein

77049.9

0.83 0.92

791.9 595

25 28

kininogen 1; alpha-2-thick proteinase inhibitor;

47883.2

0.96 0.94

626.8 453.3

29 27 bradykinin

amin precursor; alpha-albumin

69069.1

0.95 0.99

638.7 432.6

21 21

vitamin D-binding protein precursor;

52917.5

0.94 0.92

842.5 412.2

29 25 vitamin D-binding alpha-globulin

cerulaplasmin (ferroxidase);

122205.2

0.96 0.92

397.6 395.8

9 12

alpha-1-microglobulin/bikunin precursor;

36999.5

0.93 0.88

493.1 363.3

30 33 Alpha-1-microglobulin/bikunin precursor (inter-alpha-trypsin inhibitor, tight chain; protein HC); Alpha-1- microglobulin/bikunin precursor; inter-

complement component 4A

192336

0.83

355.4

6 preproprotein; acidic C4; Rodgers form of C4; C4A anaphytatoxin

complement component 4B proprotein

192797.5

0.92 0.83

490.3

6

beta-2-glycoprotein I precursor

36312.2

0.95 0.87

588.8 222.5

39 40

I factor (complement)

65788.3

0.92 0.9

507.1 319.8

28 17

hemopexin

51676.4

0.95 1.01

500.2 305.5

23 19

complement factor B preproprotein; C3

85504.8

0.92 0.92

343.4 301.8

10 13 proactivator; C3 proaccelerator; glycine-rich beta-glycoprotein; C3/C5 convartase

complement component 7 precursor

93518.2

0.93 0.91

650.7 297.6

18 15

coagulation factor XIII B subunit precursor;

75491.6

0.82 0.92

303.2 296.5

13 14 TGase

fibronectin 1 isoform 3 preproprotein;

259225.9

1.05 1.11

346.9 205.9

4 2 cold-insoluble globulin; migration-stimulating factor

complement component 1. r

80199.7

1.04 0.91

454.5 293.9

15 16

complement component 4 binding

87033.2

0.9 0.91

441.5 251.1

19 18 protein, alpha; Complement component 4-binding protein, alpha polypeptide; complement component 4-binding

alpha-2-HS-glycoprotein; Alpha-2HS-

39324.7

1.01 0.93

398.6 241

20 30 glycoprotein

plasma

B1 precursor

71369.7

0.91 1.08

531 248.4

22 18 plasma;

3, plasma;

B plasma; Fleicher factor

peptidoglycan recognition protein L precursor

67970.3

0.97 0.98

324.9 228

12 11

albumin precursor; PRO0883 protein

69366.7

0.9 0.91

491.9 220.7

18

Complement component B precursor

104844.1

1.01 0.99

300.3 171.9

10

apolipoprotein D precursor

21275.6

1 1.02

175.2 139.9

13 13

properdin P factor, complement

51278.4

0.99 1.05

151.2 116.1

10 10

complement component B. alpha

86183.2

0.83 1

340.9 168.1

19 10 polypeptide precursor

complement component 1, 3

76884.4

1.02 0.88

243.2 183.2

8 6

(plasminogen binding

22586.8

0.78 1

159.4 180.9

17 17 protein);

(plasminogen-

precursor; serum spreading

54335.7

0.95 0.96

236.2 140.2

7 7 factor; somatomedin B, complement S- protein;

apolipoprotein B precursor, apoB-100;

515862.7

1.02 0.96

135.5 155.6

1 1 apoB-16

apolipoprotein M; NG20-like protein

21253.3

0.91 0.96

98.8 147.8

12 25

attractin isoform 1; attractin-2; mahogany

158836.9

0.69 1.2

226.2 110.4

8 4 protein

coagulation factor XII precursor

67618.1

0.83 1.02

243.3 132

9 7 Hageman factor

complement component 8, beta

66947.7

0.99 0.94

80.5 127.1

11 10

alpha-2-glycoprotein 1, zinc; Alpha-2-

34258.7

1.05 0.93

108.3 121.2

7 7 glycoprotein, zinc

serine (or cysteine) proteinase inhibitor,

52602.4

0.95 1.1

165.6 115

7 7 clade C (antithrombin), member 1; antithrombin III

haptoglobin

45205.3

0.77 0.89

252.2 67.7

19 9

alpha 1B-glycoprotein

54253.5

0.94 0.91

240.7 111.4

8 8

histidine-rich glycoprotein precursor;

59675.3

0.95 0.92

258.7 105.2

10 8 histidine-proline rich glycoprotein; thrombophilia due to elevated HRG,

complement component 4 binding

28357.4

0.96 1.03

92.8 88.3

9 15 protein, beta; complement component 4- binding protein, beta polypeptide; complement component 4-binding

immunoglobulin J chain

18098.6

1.05 0.98

104.2 23

15 6

1 precursor;

-

23511.6

0.98 0.9

156.5 93.1

10 10 1 (alpha-1-acid glycoprotein-1); alpha-1- acid glycoprotein 1

complement component 2 precursor;

83287.8

1.07 1.25

64 28.2

4 3 C3/C5 convertase

alpha-2-plasmin inhibitor, alpha-2-

54595.8

0.94 0.83

87 97

6 6 antiplasmin

orosomucold 2; alpha-1-acid

23602.6

0.97 0.81

89.5 82.7

12 12 glycoprotein, type 2

protein S (alpha); Protein S, alpha

75072.5

0.94 1

164.8 95.6

8 3

clustarin isoform 1; complement-

57832.6

2.29 0.94

145.6 94.9

8 4 associated protein SP-40

complement component 9

63173.4

0.98 0.89

160.7 92.9

6 7

inter-alpha (globulin) inhibitor H1; Inter-

101402.2

0.94 0.92

31.5 87.4

2 2 alpha (globulin) inhibitor, H1 polypeptide

mannan-binding lectin serine protease I

79246.7

1 0.95

130 30.3

6 2 Isoform 1, precursor, protease, serine, 5 (mannose-binding protein-associated); manan-binding lectin serine protease-1; Re-reactive factor serine protease p100

complement component 8, gamma

22219.4

1.11 1.01

29.3 40.3

8 8 polypeptide

complement component 5

188305.3

1.01 0.97

228.6 80.6

3 2

bintinidase precursor

61132.9

0.94 0.9

145.5 42.5

7 3

PREDICTED:

to

85603.6

1.02 1.01

40.7 78.6

2 7 Carboxypeptidase N 83 KDa chain (Carboxypeptidase N regulatory subunit)

apolipoprotein A-II precursor

11175

1 0.75

114.1 61.7

20 20

serine (or cysteine) proteinase inhibitor,

47850.9

0.88 0.87

17.2 45.1

2 2 clude A, member 3 precurseor, alpha-1- antichymotrypsin; antichymotrydain

haplogtobin-reisted protein; Haptoglobin-

39029.6

0.86

58.5

10 related locus

coagulation factor V precursor; labile

251719.4

0.82 0.87

56.6 5.9

1 1 factor, factor V Laiden

Insulin-like growth factor binding protein,

66035

0.92 0.97

77.3 39.8

3 2 acid labile submit; INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN COMPLEX ACID LABLE CHAIN

M factor (complement)-like 3; factor H-

30650.7

1 1.08

108.7 40

11 7 ralated gene 2

H factor (complement)-like 1

37881.8

0.91 0.95

143 61.5

12 8

sex hormone-binding globulin; Sex

43778.2

0.99 1.01

26 60.3

2 2 hormone-binding globulin (androgen binding protein)

keratin 1; Keratin-1; cytokeratin 1; hair

68066.7

N/A 1.08

10.6 57.6

4 4 alpha protein

apolipoprotein E precursor;

36154.1

1.08 1.01

37.5 57.3

3 3 apolipoprotein E3

Insulin-like growth factor binding protein

31660.2

1.02 1.1

33.4 54.4

6 12

cysteine-rich secretory protein 3; specific

27630.3

0.77 0.87

45 44.2

8 3 granule protein (28 kDa); cysteine-rich secretory protein-3

HGF activator preproprotein

70661.8

0.88 1.14

18.3 28.8

1 1

gelsolin isoform a

85697.6

0.81 0.9

105.9 38.4

1 1

apolipoprotein F precursor

36399.6

0.86 1.49

41

3

CD5 antigen-like (scavenger receptor

38087.8

1.02 1.03

140.5 46.7

14 6 cysteine rich family);

; apoptosis Inhibitor B

hyaluronan binding protein 2; hyaluronic

52671.7

1.04 0.94

145.7 36.6

6 2 acid binding protein 2, hepatocyte growth factor activator-like protein; plasma hyaluronan binding protein; factor VII activating protein; hyaluronan-binding protein 2

protein C (inactivator of coagulation

52071.3

1.2 1.02

59.5 45.7

5 9 factore Va and VIIIa)

feluin B; feluin-like protein

42094

0.96 0.75

84.3 44.6

2 2

extracellular matrix protein 1 isoform 1

60704.1

1.1 0.89

21.7 43.1

1 3 precursor; secretory component p85 precursor; secretory component p85

lumican

38429

1 1

59.1 22.2

4 4

protein Z, vitamin K-dependent plasma

44743.9

0.97 0.85

85.3 41.2

7 3 glycoprotein

CD44 antigen isoform 1 precursor, cell

81537.6

0.94

40.7

1 surface glycoprotein CD44; Lutheran inhibitor, dominant; homing function and Indian blood group system; monoclonal antibody A3D8; antigen gp90 homing receptor, CDW44 antigen; phagocylic glycoprotein I; extracellular

serine (or cysteine) proteinase inhibitor,

46312.2

2.11 0.91

26.9

2 clade F (alpha-2 antiplasmin, pigment epithelium derived factor, member 1; pigment epithelium-derived factor

platelet glycoprotein Ib alpha polypeptide

68955.3

1.13 0.95

32.6 39.5

1 5 precursor, platelet membrane glycoprotein 1b-alpha subunit1

macrophage stimulating 1 (hepatocyte

80319.9

0.99 1.01

27.8 39.4

0 1 growth factor-like)

complement component 1, q

26016.8

0.85 0.91

subcomponent, alpha polypeptide precursor, complement component C1q.

programmed cell death B isoform 1;

66900.5

N/A

38

1 apoptosis-inducing factor

plasma glutathione peroxidase 3

25402.3

1 0.9

58.2

6

plasma carboxypeptidase B2 isoform a

48442.2

0.93 0.84

101.4 37.6

8 3 praproprotein; carboxypeptidase U; thrombin-activatable fibrinolysis inhibitor; carboxypeptidase B-like protein; thrombin-activable fibrinolysis inhibitor

complement component 1, q

25703.7

1.03 0.68

11.9 37.5

3 3 subcomponent, beta polypeptide precursor; complement component C1q.

heparin cofactor II

57098.6

1.24

coagulation factor XIII A1 subunit

83233.3

0.81

precursor, Coagulation factor XIII, A polypeptide; TGase

complement component 1 inhibitor,

55153.2

0.93 0.91

60.4 38.4

2 2 precursor

serine (

cysteine) proteinase inhibitor,

50707

0.8 N/A

25.4

2 clade A (alpha-1 antiproteinase,

), member 10; protein Z- dependent protease inhibitor precursor; protein Z-dependent protease inhibitor precursor

cultin 1

89678.5

1.02 0.97

27.6

1

coagulation factor X precursor

54731.7

1.02 0.89

95.9 10.2

7 0 prothrombinase; factor Xa

carboxypeptidase N, polypeptide 1, 50 kD

52286.2

1.02 1.01

18.5

2 precursor

inter-alpha (globulin) inhibitor H2; inter-

105713.9

0.91 0.31

15.8 31.3

0 2 alpha (globulin) inhibitor, H2 polypeptide

coagulation factor IX; Coagulation factor

51778.4

1.05 1.82

22.5

2 IX (plasma thromboplastic component); Factor 9; Factor IX; Christmas factor

hypothetical protein DKFZp434F1726

213146

2.48 0.85

10.4

0 isoform 1

SMC6 protein

126325.6

0.93 0.94

26.6

1

ring finger protein 130;

protein;

46404.9

0.93 4.27

16.6 17

1 1 g1-related zinc finger protein

arylsulfatase B isoform 1 precursor; N

59687.2

0.99 0.68

acetylgalactosamine-4-sulfatase

galactin 3 binding protein; L3 antigen

65331

2.19

27.8

2 Mac-2-binding protein; serum protein

fibrinogen, beta chain preproprotein

55902.1

0.9 N/A

23.7 27.2

1 1

CD14 antigen precursor

40076.2

0.96 1.09

20.3 18

4 2

paraxonase 1; Paraxonase

39731.3

0.94 0.95

29.9 26.1

3 3

metalithionein 1A

5133.3

N/A

cholesteryl ester transfer protein, plasma

54770.2

0.99 1.14

17.5 23.4

2 2 precursor

hypothetical protein FLJ34064

97726.3

0.73 N/A

23.1

0

Inter-alpha (globulin) inhibitor H4 (plasma

103357.4

1.12

Kallikrein-sensitive glycoprotein); Inter- alpha (globulin) inhibitor. H polypeptide- like 1; inter-alpha (globulin) inhibitor, H4 polypeptide

mitogen-activated protein kinase kinase

81296.4

2 21 10.22

12.8

2 kinase kinase 1; hematopoietic proganitor kinase 1

calcium binding protein 39-like

33694.2

1

22.9

3

a disintegrin-like and metalloprotease

214506.1

0.65 1.17

45.1 22.8

1 0 (maprolysin type) with thrombospondin type 1 motif, 20 isoform 1; a disintegrin- like and metalloprotease with thrombospondin type 1 motifs 20

ephrin receptor EphB4 precursor;

108270.2

0.98 0.29

13.7 22

0 0 hapatonia transmembrane kinase

PREDICTED: chromosome 8 open

37270.1

0.95

21.8

1 reading frame 4

solute carrier family 37 member 1;

57848.1

0.99

21.7

1 glycerol-3-phosphate permease

zinc finger protein 568

43735.1

1.03 1.35

12.4

1

gofgi autoantigen, golgin subfamily a, 4;

261140.2

0.88 0.9

12.3 20.4

0 0 gofgin-245; trans-Golgi p230; 256 kDa golgin: 72.1 protein; golgin-240

hypothetical protein BC008322

34854.5

2.11 0.9

20

4

L-plastin; Lymphocyte cytomotic protein-1

70289.3

0.75 0.95

19.3

2 (plasmin); plastin 2

serine (or cysteine) proteinase inhibitor,

48738.6

1 0.88

20.3

2 clade A (alpha-1 antiproteinase, antitrypsin), member 1; protease inhibitor 1 (anti-elastase), alpha-1-antitrypsin

leucine-rich repeat-containing G protein-

99266.8

1.01 0.95

27.8

0 coupled receptor 6

plasma coagulation factor XI precursor

70109.1

1 1.09

53.2

8 isoform a; plasma thromboplastin antecadent

serine (or cysteine) proteinase inhibitor,

46542

1.04 0.89

12.8

1 clade A (alpha-1 antiproteinase, antitrypsin), member 4; protease inhibitor 4 (kellistatin)

transthyretin; prealbumin

15887

1 1.35

27.8 14.3

4 4

complement component 1, q

25729.6

0.95 1.04

49.6 15.3

4 4 subcomponent, gamma polypeptide; complement component C1q, C chain

ubiquitin protein ligase E3 component n-

200210.8

1.17 1.67

12.5

0 recognin 1: ubiquitin ligase E3 alpha-I

excision repair cross-complementing

89277.7

0.75 1.09

24.8 14.9

1 1 rodent repair deficiency, complementation group 3; xeroderma pigmentosum, complementation group 5

PREDICTED; similar to KIAA0446

296001.2

0.84 0.74

19.9

0

T1 protein

139422.9

0.94 1.41

13.3 13.7

0 0

retinoblastoma-associated factor 800

573901

0.6 N/A

58.8

0

PREDICTED: KIAA1083 protein

188211.8

N/A 0.85

20.5 13

1 0

PREDICTED: hypothetical protein

29364.1

N/A 0.99

27.8 11.5

3 2

zinc finger protein ZNF-U89274: zinc

119382.6

N/A N/A

15.8 11.2

0 0 finger protein

hypothetical protein FLJ36728

72363.7

0.82 1.19

25.1 10.7

2 1

pregnancy zone protein; Pregnancy zone

163835.9

1.02

protein

altractin isoform 3; altractin-2; mahogany

133701.6

0.9

protein

mannan-binding lectin serine protease 1

81860.3

0.97

135

6 isoform 2, precursor, protease, serine, 5 (mannose-binding protein-associated); manan-binding lectin serine protease-1; Re-reactive factor serine protease p100

cartilage oligomeric matrix protein

82880.5

1.22

102.7

7 precursor; epiphyseal dysplasia, multiple 1; pseudoachondroplasia (epiphyseal dysplasia 1, multiple); cartilage oligomeric matrix protein(pseudoachondroplasia, epiphyseal dysplasia 1, multiple);

fibulln 1 isoform C precursor

74461.9

0.78

48.3

2

lipoprotein. Lp(a); Apolipoprotein Lp(a);

501319.1

1

92.2

1 antiangiogenic AK38 protein

von Willebrand factor precursor:

309298.6

1.09

84.3

1 Coagulation factor VIII VWF (von Willebrand factor)

low density lipoprotein-related protein 1;

504575.3

0.98

42.9

0 alpha-2-macroglobulin receptor

procollagen C-endopeptidase enhancer;

47946.4

1.38

84.2

6 procollagen, type 1, COOH-terminal proteinase enhancer

plasminogen-related protein B; type B

10970.5

0.99

plasminogen-related gene

PREDICTED: kelch repeat and BTB

631761.2

1.15

79.4

0 (POZ) domain containing 9

stabilin 2; CD44-like precursor FELL;

276994.1

N/A

14

0 hyaluronan receptor for endocytosis

low density lipoprotein-related protein 1B;

515398.8

0.62

66.4

0 low density lipoprotein receptor related protein-deleted in tumor

KIAA1404 protein

220228.8

1.73

46.1

1

selectin L; lymph node horning receptor,

42187.1

0.87

58.3

0 lymphocyte adhesion molecule 1

dynein, axonernal, heavy polpeptide 8

514819.9

1.04

58.3

1

FC fragment of IgG binding protein; IgG

572096.7

1.24

11

0 Fc binding protein

v-ski sarcoma viral oncogene homolg;

80005.1

1.35

52.8

4 Avian sarcoma viral (v-ski) oncogene homolog; v-ski avian sarcoma viral oncogene homolog

PREDICTED: similar to hypothetical

39317

0.82

49.1

5

GREB1 protein Isoform a; gene regulated

216467.3

1

46

1 by cetrogen in breast cancer protein

hypothetical protein FLJ13908

28645.1

N/A

a disintegrin and metlioproteinase with

216491.2

0.26

45.7

1 thrombospondin motifs 9 isoform 1 preproprotein

DNA (cytosine-5-)-methyltransferase 1;

183165.2

0.6

45.5

0 DNA methyltransferase; DNA methyltransferase 1

hypothetical protein LOC129607

32645.7

0.72

44.8

6

ubiquitous tetratricopeptide containing

109658

0.66

44.2

2 protein RoXaN; Rotavirus ‘X’ associated non-structural protein

scavenger receptor Cysteine-rich type 1

158257.1

N/A

44.1

3 protein M160 precursor; CD163 antigen

integrin beta chain, beta 2 precursor,

84790.8

1.48

43.9

3 integrin, beta-2 (antigen CD18 (p95), lymphocyte function-associated; cell surface adhesion glycoprotein (LFA- 1/CR3/P150, 959 beta subunit precursor)

fibrinogen, gamma chain isoform

49481.5

1.1

19.2

4 gamma-A precursor

PREDICTED: hypothetical protein

18929.2

N/A

42

7

PREDICTED: similar to RIKEN cDNA

142106.7

1.03

41.5

0 5430400H23

protein kinase, lysine deficient 1; kinase

260765.7

1.18

40.1

0 delicient protein

transmembrane protease, serine 6;

90000.1

0.89

40

2 membrane-bound mosaic sarine proteinase mairplase-2; transmembrane serine protease 8; type II transmembrane serine protease 6

zinc finger protein 560

91135

1.01

39.3

1

factor H-related protein 5

64419.4

0.43

38.8

1

prenylcysteine oxidase 1; prenylcysteine

58700.2

0.98

36.8

1 lyase

zinc finger protein 625

34746.3

2.17

17.1

4

hypothetical protein FLJ32954

65228

0.4

38.2

2

similar to Hypothetical zinc finger protein

63463.3

N/A

KIAA1559

guarrytale binding protein 4-like

72525.3

N/A

37.9

3

zinc finger protein 521; early

147886.1

N/A

hematopalatic zinc finger

PREDICTED: chromosome 20 open

90734.9

1.36

reading frame 82

smooth muscle myosin heavy chain 11

223577.3

0.8

35.1

1 isoform SM2

hypothetical protein SB153 isoform 2

33050.1

1.43

phospholinositide-3-kinase, class 2, alpha

190737.7

N/A

35.6

1 polypeptide; C2-containing phosphalidylinositol kinase; PI3K-

immunoglobulin superfamily, member 10

290837.9

0.81

myotonic dyatrophy protein kinase like

172518.3

0.94

21.8

2 protein; protein kinase

complement factor D preproprotein;

27032.9

0.82

22.7

8 adipsin; propendin factor D; C3

zinc finger protein 592

137555.2

1.13

34.4

2

“NOV1”

24619.7

1.53

34.1

5

multiple inositol polyphosphate histidine

55051.2

0.93

34.1

1 phosphatase 1; multiple inositol polyphosphate phosphatase 2; multiple inositol polyphosphate phosphatase 1

KIAA1985 protein

144776.6

2.04

34.1

1

insulin-like growth factor 2 (somatomedin

20140.3

0.8

38.7

5 A); somatomedin A

WINS1 protein isoform 2

49325

0.93

33.7

1

v-lof Hardy-Zuclerman 4 feline sarcoma

109664.6

2

33.4

2 viral oncogene homolog precursor

hyrosine kinase 2

133665.9

N/A

KIAA1729 protein

119531.2

N/A

zinc finger protein 261

152379.1

1.25

zinc finger protein KIAA0961

61557.5

N/A

PREDICTED: zinc finger CCCH type

108458.6

1.12

32

3 domain containing 5

mannose receptor, C type 2; andocytic

186655.4

2.98

12.8

1 receptor (macrophage mannose receptor family); urokinase plasminogen activator receptor-associated protein

zinc finger, SWTM domain containlng 4

110138

N/A

31.7

1

PREDICTED: hypothetical protein

11479

0.88

31.4

8

PREDICTED: hypothetical protein

22383.2

N/A

31.3

6

complement factor H-related 4

37325

0.88

31.1

3

spectrin, beta, non-erythrocylic 5: beta V

416835.1

N/A

spectrin

KIAAD676 protein isoform

140624.7

0.82

aquaporin 3

31543.8

N/A

somaphorin 4D; sama domain,

96207.9

1.08

29.6

0 Immunoglobulin domain (lg). transmembrane domain (TM) and short cytoplasmic domain, 4D

hypothetical protein MGC34032

81743.3

0.94

PREDICTED: similar to KIAA0033

70600.3

N/A

20.6

1

csln

504587.3

0.38

28.8

0

wingless-type MMTV integration site

39000.8

N/A

28.4

2 family. member 9B precursor. wingless- type MMTV integration site family,

tripartite motif protein 15 Isoform beta

12301.1

0.88

11.2

8

proteasome alpha 7 subunit isoform 1;

27586.8

1.76

proteasome subunit RC8-1; proteasome subunit XAPC7

winglass-type MMTV Integration site

46444.3

0.66

27

5 family, member 10A precursor

gp130-like monocyte receptor; soluble

82953.8

0.74

27

1 type I cytokine receptor CRL3; GP130 like receptor

5100 calcium-binding protein A8;

10834.5

0.71

calgranulin A; cysile librosis antigen; 8100 calcium-binding protein A8

extracellular matrix protein 2

79789.3

N/A

kinase insert domain receptor (a type III

151526.8

2.03

receptor tyrosine kinase); Kinase insert domain receptor

PR domain containing 11; PR-domain

57862.9

0.77

containing protein 11

hypothetical protein FLJ14936

37476.5

N/A

zinc finger protein 177

36473.1

1.12

26

3

neuroplastoma-amplified protein

268571.3

0.96

gonadolrapin inducible transcription

88214

0.97

repressor 1

a disintegrin-like and metalloprotease

136167.1

N/A

25.4

1 (reprolysin type) with thrombospondin type 1 motif, 18 isoform 1 preproprotein, a disintegrin-like and metalloprotease (reprolysin type) with thromboepondin type 1 motiff, 21

thromboepondin type | domain-containing

94683.9

0.86

25.4

1 1 isoform 1; 4833423O18Rlk; transmembrane molecule with thromboepondin molecule;

tarsh protein

118642

0.88

24.7

1

rhoinbold, veinlet-like 4; ventrifold

45244.6

0.33

25.1

4 transmembrane protein

glutamate recaptor KA1 precursor,

107245.5

N/A

excitalory amino acid receptor 1

splicing factor, arginine-serine-rich 5

12626

0.76

24.8

9

PREDICTED: similar to Afaxin-1

70403.9

N/A

24.8

2 (Spinocarebeller alaxis type 1 protein homolog)

heat shock 90 kDa protein 1, alpha; heat

64673.7

0.91

24.7

0 shock 90 kDa protein 1, alpha

PREDICTED: similar to carbonic

70702.3

N/A

18

2 anhydrase VA, mitochondrial precursor; carbonic anhydrase V, mitochondrial, carbonic dehydralase

PREDICTED: myosin VB

266807.1

3.2

24.5

0

hypothetical protein FLJ14788

51893.9

N/A

24.2

3

hypothetical protein DT1P1A10

20894

N/A

24.2

4

signal transducer and activator of

90647

N/A

24

0 transcription 6A

thyroid peroxidase isoform a;

102982.7

1.34

12.2

1 thyroperoxidase: thyroid microsomal antigen

DEAH (Asp-Glu-His) box polypeptide

78874.1

N/A

33; DEADH (Asp-Glu-Ala-Asp/His) box polypeptide 33

protein phosperilase 5, catalytic subunit

58878.6

N/A

PREDICTED; KIAA1447 protein

269382.4

0.81

HLA-B associated transcript 5; HLA-B

83243.8

1.06

23.8

2 associated transcript-5; BAT5 protein

interferon-alpha receptor 1 precursor;

63525.3

N/A

alpha-type antiviral protein; beta-type antiviral protein; interferon-beta receptor 1; interferon-alpha/beta receptor alpha

PREDICTED: similar to 33 kDa protein

40917.2

0.78

23.7

1

neuralized-like protein 2; neuralized-like

31689.6

N/A

2; chromosome 20 open reading frame

solute carrier family 29 (nucleoside

58114.7

0.96

23.6

3 transporters), member 4; aquilibrative nucleoside transporter 4

PREDICTED: DNA2 DNA replication

129681.5

0.57

12.4

1 helicase 2-like

vanin 1 precursor, Vannin 1;

67023.7

0.67

23.5

2

neuraxin 1 isoform alpha precursor;

161882.9

N/A

23.5

1 neuraxin 1

coronin, actin binding protein, 1A;

51026.3

N/A

23.5

2 coronin, actin-binding, 1A; coronin, actin- binding protein, 1A; coronin-1

similar to 60S ribosomal protein L10(QM

24626.9

N/A

23.3

4 protein homolog)

chromosome 20 open reading frame 42;

77408.8

N/A

23.2

0 UNC-112 related protein 1; kindlin 1; kindlerin

PREDICTED: bicaudal C homolog 1

53276.3

N/A

23.2

3

PREDICTED: similar to Charot-Layden

16189.4

N/A

17.3

6 crystal protein; osinophil lysophospholpase; lysolacthin acylhydradase; gatactin-10

hypothetical protein FLJ10863

16473.8

N/A

hypothetical protein XP_376795

19258.2

0.88

22.9

7

PREDICTED: similar to ankylin repeat

60218.7

0.82

domain 20A

Interleukin 19 isoform 2 precursor;

20451.8

N/A

22.6

5 melanoma differentiation associated protein-like protein

axostosin 1

86254.8

N/A

solute carrier organic anion transporter

77193.3

0.65

22.7

0 family member 4A1; solute carrier family 21 member 12; organic anion transporting polypeptide E; sodium- independent organic anion transporter E; organic anion transporter polypeptide- related protein 1; colon organi

PREDICTED: similar to P38IP protein

93655

N/A

22.6

0

connector enhancer ol kinase

117534.5

N/A

suppressor of Ras 2; connector enhancer

Inonitol 1,4,5-triphosphate receptor, type

306773.3

0.99

21.5

0

surfactant, pulmonary-associated protein

26169.3

0.95

22.6

2 AZ

PREDICTED similar to Dual specifity

38366.5

0.98

22.3

2 protein phosphatase 13 (Tests-and skelatal-muscle-specific DSP)

mitochondrial ribosomal protein L45

35242.8

0.98

22.1

4

trypothetical protein FLJ20438

43483

N/A

sarF domain containing kinase 6

55896.7

0.84

21.9

3

heparan sullfate 5-O-sulfotransferance;

47075.8

N/A

21.9

2 heparan-sulfate 6-sulfotransferase

rab-related GTP-binding protein

25006.7

0.99

21.8

2

helecase/primase complex protein

27664.6

0.81

similar to S. cereviase SSM4

102545.2

N/A

21.8

0

PREDICTED: similar to Vascular

47909

N/A

endothelial growth factor receptor 1 precursor (VEGFR-1) (Vascular permeability factor receptor) (Tyroasine- protein kinase receptor FLT)(Flt-1) (Tyrosine-protein kinase FRT) (Fma-like

zinc finger protein 639; Kruppel-like; zinc

58054.4

N/A

finger protein ANC 2H01

Bent-like 1; BarH (Drosophila)-like 1

35074.5

N/A

21.7

5

calhepsin S preproprotein

37495.7

1.02

21.7

2

sarco/endcoplasmic reticulum Ca2+-

109258.2

N/A

ATPase isoform a; ATPase, Ca(2+)- transporting, ubiquitious; sarcoplasmic/endoplasmic reticulum calcium ATPase 3; SR Ca(2+)-ATPase 3; calcium pump 3; adenosine triphosphatase, calcium;

glypican 5

53707

N/A

leucine rich repeat transmembrance

59078.2

0.62

21.5

2 neuronal 2

solute carrier family 12, member 5; solute

78224.8

0.94

13.9

1 carrier family 12 (sodium/potassium/chloride transporters), member 8; cation-chloride

zinc finger protein 228

105076.4

N/A

21.4

1

PREDICTED: similar to Mucin 1

93058.4

N/A

21.4

0 Precursor (MUC-1) (Polymorphic epthelial mucin) (PEM) (PEMT) (Eplstalin) (Tumer-associated mucin) (Carcinoma-associated mucin) (Tumor- associated epithelial membrane antigen) (EMA) (H23AG) (Peanul-reactive urinary

tankyrase, TRF1-interacting ankyrin-

142011.5

1.38

21.3

0 related ADP-ribose polymerase

1-acylglycerol-3-phosphate O-

43381

0.89

21.3

1 acyltransferase 3; lysophosphatidic acid acyltransferase-gamma1; 1-acyl-sn- glycerol-3-phosphate acyltransferase gamma; 1-AGP acyltransferase 3

ATP-binding cassette, sub-family C,

149540.8

0.57

21.3

0 member 4; canallcular multispecific organic anion transporter (ABC

brain glycogen phosphorylase; glycogen

96696

N/A

21.3

1 phosphorylase B

potassium channel tetramerisation

88984

N/A

14.6

1 domain containing 3; NY-REN-45 antigen

PREDICTED: similar to ODZ3

21695

0.99

21.3

8

potassium voltage-gated channel, Shal-

70536.5

N/A

related subfamily, member 2; voltage- sensitive potassium channel; voltage- gated potassium channel Kv4.2

PlggyBac transpossble element derived 2

68011.4

0.68

21.1

2

hypothetical protein FLI90430

61740.9

0.79

11.8

1

alyl hydrocarbon receptor

96147.4

0.91

13.5

0

SH3 and cysleine rich domain; arc

44553.5

N/A

21

2 homology three (SH3) and cysleine rich domain

phosphatidylinoskol-4-phosphate 5-

61036.3

0.49

20.9

1 kinase, type I, beta

laucine-rich repeats and immumoglobulin-

76433.8

N/A

like domains 4

tripartile motif-containing 3g isoform 1;

59990.4

N/A

ring finger protein 23; testis-abundent finger protein

hypothetical protein FLJ23153

31814.1

1.02

20.8

3

dihydropyrimidinase-like 4

61905.7

N/A

growth hormone 2 isoform 3; hGH-V

27101.2

1.05

20.6

3 placental-specific growth hormone; placenta-specific growth hormone

KIAA0218 gene product

85023.1

0.89

12.8

2

olfactory receptor, family 6, subfamily C,

35116.5

1.09

20.5

4 member 76

coli division cycle 2-like 5 isoform 1;

164970.3

N/A

20.3

0 CDC2-related protein kinase 5

hypothetical protein FLJ38808

64517.4

N/A

phosphofurin acidic cluster sorting

104898.4

N/A

protein 1; cytosolic sorting protein PAC8-

sushi domain containing 2; Sushi domain

90207.7

N/A

20.3

1 (SCR repeat) containing

myosin XV; unconvertional myosin-15

395219.5

N/A

20.2

0

chamokine (C—C motif) receptor 8;

40844.4

N/A

20.2

2 chamokine (C—C) receptor 8; chamokine (C—C) receptor-like 2; CC-chamokine receptor chamr1

PERQ amino acid rich with GYF domain

89740.9

N/A

20.2

1 1; postmelotic segregation Increased 2- like 12; Grb10 interacting GYF protein 1

vacuolar protein sorting 29 isoform 1;

20505.7

0.72

20.2

3 vacuolar sorting protein VPS29/PEP11; vacuolar protein sorting 29 (yeast homolog); retromer protein; x007 protein

hypothetical protein MGC45888

31064.9

N/A

20.1

5

D site of albumin promoter (albumin D-

34348.9

0.57

box) binding protein; D site of albumin promoter binding protein

transducin-like enhancer protein 2;

79341

0.8

20.1

2 transducin-like enhancer of split 2; enhancer of split groucho 2; transducin- like enhancer of split 2, homolog of Drosophila E(sp1)

hypothetical gene MGC 16309

36338.8

0.61

splicing factor, arginineserine-rich 1

27744.6

1.79

(splicing factor 2, alternate splicing

super conserved receptor expressed in

41481.4

N/A

20

5 brain 3

hypothetical protein XP, 211108

10737.5

1.41

20

17

According to these results, 158 proteins could be identified and comparatively quantified when analyzing this fraction (SCX 50 fraction) on ABI-4700 and selecting, at Rank 1, peptides having a Mascot score of 30 or higher, and about 286 proteins were identified and comparatively quantified when selecting peptides a Peptide Score of 20 or higher. When the SCX 50 fraction was analyzed similarly in the C18-nanoLC/Q-Star system, 119 proteins could be identified and quantified in the case of selecting, at Rank 1, peptides having a Peptide Score of 20 or higher. In addition, the ratios of H/L-chain labeling (comparative quantification values) of most proteins were approximately 1, and thus it appears that the comparative quantification method according to the present improvement can be satisfactory.

By comparing top 119 proteins on ABI-4700 and top 119 proteins on Q-Star, 80 proteins were common in both, 39 proteins were determined and quantified only on Q-Star, 39 proteins only on ABI-4700, and a total of 158 proteins on either of the instruments (FIG. 2). When selecting a score of 20 or higher on ABI-4700 and on Q-Star, 94 proteins were common in both, 25 proteins were identified only on Q-Star, 192 proteins only on ABI-4700, and a total of 311 proteins on either of the instruments (FIG. 3).

It turns out from the above-described results that by using the method according to the present invention, a plurality of small-amount proteins in serum can be identified and comparatively quantified.

Comparative Example Identification and Quantification of Serum Proteins by Conventional Method

According to the routine procedure, serum (in which the six major proteins, including albumin, had been removed) was reacted with a cICAT reagent, the resultant labeled proteins were digested with trypsin, and the reaction solution containing the trypsin digestion products was loaded onto SCX column chromatography to thoroughly remove reagent-derived substances and others, followed by fractionating the peptide fraction into 50 sub-fractions with a salt concentration gradient method. The obtained sub-fractions were further loaded onto an avidin affinity column to specifically purify labeled peptides containing biotin. The labeled peptides containing biotin were treated with TFA to cleave the biotin segment and others, followed by evaporation to dryness. The obtained samples were subjected to measurements on a mass spectrometer to identify and quantify serum proteins, whereby major serum proteins (30 to 50 proteins, Mascot Scores of 20 or higher) could be identified and quantified, small-amount proteins could hardly be identified. From the results of the investigation as to this cause, it turned out that each of the fractionated samples after the above-described TFA treatment contains biotin at a much larger amount than the equivalent amount of biotin derived from the labeled peptides containing biotin. 

1. A method for the analysis of differential expression of proteins employing an isotope label, characterized by cleaving a tag from peptides labeled with an ICAT reagent containing a cleavable tag, separating and purifying the resultant labeled peptides, and performing an analysis in mass spectrometry.
 2. The method according to claim 1, wherein the step of separation and purification is carried out using column chromatography and wherein the removal of the tag and others the separation and purification of the cICAT-labeled peptides are carried out concurrently.
 3. The method according to claim 1 or 2, wherein the tag is biotin.
 4. The method according to any one of claims 1 to 3, wherein the peptides are derived from a serum protein.
 5. A system for the analysis of differential expression of small-amount proteins in a sample, characterized by employing a method according to any one of claims 1 to
 3. 6. The system according to claim 5, wherein the sample is a serum sample. 